Abstract
Benzoyl-CoA reductase catalyzes the two-electron transfer from a reduced ferredoxin to the aromatic ring of benzoyl-CoA; this reaction is coupled to stoichiometrical ATP hydrolysis. A very low reduction potential (less than -1 V) is required for the first electron transfer to the aromatic ring. In this work the nature of the redox centers of purified benzoyl-CoA reductase from Thauera aromatica was studied by EPR and Mössbauer spectroscopy. The results obtained indicated the presence of three [4Fe-4S] clusters. Redox titration studies revealed that the reduction potentials of all three clusters were below -500 mV. The previously reported S = 7/2 state of the enzyme during benzoyl-CoA-independent ATPase activity (Boll, M., Albracht, S. J. P., and Fuchs, G. (1997) Eur. J. Biochem. 244, 840-851) was confirmed by Mössbauer spectroscopy. Inactivation by oxygen was associated with the irreversible conversion of part of the [4Fe-4S] clusters to [3Fe-4S] clusters. Acetylene stimulated the benzoyl-CoA-independent ATPase activity and induced novel EPR signals with g(av) >2. The presence of simple cubane clusters in benzoyl-CoA reductase as the sole redox-active metal centers demonstrates novel aspects of [4Fe-4S] clusters since they adopt the role of elemental sodium or lithium which are used as electron donors in the analogous chemical Birch reduction of aromatic rings.
Highlights
For many decades it appeared that the biological metabolism of aromatic compounds was restricted to an aerobic metabolism
The nature of the iron-sulfur centers of benzoylCoA reductase (BCR) was studied by Mossbauer and electron paramagnetic resonance (EPR) spectroscopy
High 57Fe enrichment was achieved as estimated from the resonance absorption effect of about 9% for each subspectrum of the oxidized sample, which corresponds to about 11 mM 57Fe
Summary
For many decades it appeared that the biological metabolism of aromatic compounds was restricted to an aerobic metabolism. A radical mechanism, corresponding to the Birch reduction, of alternate one-electron and oneproton transfer steps to the aromatic has been proposed for the BCR reaction [5, 6]. BCR hydrolyzes MgATP in the absence of a reducible substrate [3] This uncoupled ATPase activity was only observed in the dithionite-reduced and not in the thionine-oxidized state of the enzyme [8]. A shift from interacting to non-interacting [4Fe-4S]ϩ1 clusters was detected These observations allowed the presentation of a first model for the catalytic cycle of the BCR reaction. This model included an ATP-driven electron activation as a result of conformational changes affecting a reduced, EPRactive iron-sulfur cluster [8]. The ATP-dependent switch from the S ϭ 1/2 to the S ϭ 7/2 state was confirmed by Mossbauer spectroscopy and could clearly be assigned to a [4Fe-4S] cluster
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